An agglutination assay or agglutination reaction is an important and widely known technique in the biological and chemical sciences for detecting and/or quantifying chemical reagents. Agglutination assays have been used in a wide variety of applications to detect biological or chemical entities. For example, agglutination assays have been used to detect bacteria such as Mycobacteria leprae (leprosy) and tuberculosis, Vibrio cholera (cholera), Yersinia enterolytica, Borrelia burgdorferi (Lyme disease), and other bacteria. Agglutination tests have also been used to detect viral pathogens including HIV, Herpes simplex, cytomegalovirus, influenza and other viruses. Agglutination assays have also been used to determine blood type, and to detect antibodies or antigens in a variety of biological fluids such as saliva, urine, blood or serum. Applications of various embodiments of detection of agglutination by optical density measurement discussed in this application include detection of agglutination in any agglutination assay, without limitation or modification to any specific goals of the agglutination assay itself, such as to detect a particular virus or other entity.
In a type of agglutination assay, particles are suspended in a reaction medium and one or more reagents are added. The particles may be synthetic or naturally occurring, for example, cells. In the presence of a sufficient quantity of a specific agglutination agent, under conditions suitable for agglutination, the agglutination agent will cause clumping of the particles and one or more complexes of particles will form. These clumps may be of such a structure than they prevent particles from settling under the influence of gravity. For example, if the container in which the agglutination reaction is contained has a constrictions, such as, a throat or smaller diameter section, the clumping of particles into one or more complexes inhibits the clumped particles from settling through the constriction whereas if agglutination does not occur, the particles move through the constriction to settle at a bottom of the container. Throughout this application, the process of forming of one or more complexes that prevent or inhibit settling of particles under the influence of gravity or other force will be referred to as agglutination.
An agglutination assay has long been used as a method for detecting the presence of biological agents in a sample. As a specific example, in a hemagglutination (HA) assay, erythrocytes can be used as the particles and the agglutinating agent can be influenza virus particles. In such an HA assay, the influenza particles, each having multiple receptors with the ability to bind to erythrocytes, serve as cross-linking agents and generate cross-linkages between erythrocytes. If the concentration of viruses in a sample is sufficiently high, under conditions suitable for agglutination, the erythrocytes will agglutinate.
The HA assay is commonly used as a semi-quantitative method to determine the concentration of virus (titer) in an unknown sample. To measure the titer of a virus in a sample, serial dilutions of the sample are prepared and combined with a standard concentration of erythrocytes in a reservoir under conditions that are suitable to allow agglutination to occur. At sufficiently high concentrations of virus relative to the concentration of erythrocytes, the virus will cause agglutination of erythrocytes, while at sufficiently low concentrations of the virus agglutination will not occur. By determining the highest dilution at which agglutination occurs for a known concentration of erythrocytes, an estimate of the concentration of the virus can be obtained.
A variation of the HA assay is the hemagglutination inhibition (HAI) assay. The HAI assay is widely used as a semi-quantitative method for determining the concentration of specific influenza antibodies in an unknown sample. As an example, the unknown sample in the HAI assay may be a blood serum sample from a human subject that has been vaccinated by exposure to a specific type of influenza hemagglutinin, and the concentration of antibodies to the specific type of influenza hemagglutinin in the serum sample may indicate the efficacy of the vaccine in generating immunity to the specific type of influenza virus. In the HAI assay, serial dilutions of the unknown sample are prepared and mixed with a fixed concentration of erythrocytes and virus particles. At sufficiently high antibody concentrations, antibodies with binding affinity for the specific virus will bind to the virus particles and block their ability to cross-link erythrocytes, thus preventing agglutination. At sufficiently low antibody concentrations (or in the absence of antibodies with binding affinity to the specific virus) the virus will cause agglutination of erythrocytes to occur. The highest dilution factor of the unknown sample that prevents agglutination is the HAI titer value, and serves as a metric representing the concentration of the antibodies with binding affinity for the specific virus in the unknown sample.
The HA and the HAI assays are widely used in the influenza vaccine industry to estimate the concentration of both viruses and virus-specific antibodies in unknown samples. Typically, the assay is used to analyze multiple unknown samples in parallel in a 96-well plate format, together with a standard (known) sample for calibration and a set of appropriate control tests. To improve the accuracy of the resulting measurements, serially-diluted samples are often tested in duplicate or triplicate at each concentration. Thus, for each unknown sample a large number of wells must be evaluated for the presence or absence of agglutination.
Detection of agglutination of erythrocytes or other particles in a reaction reservoir in an agglutination assay is typically accomplished by examining the bottom of the reaction vessel after a period of time sufficient to allow free particles to settle to the bottom. The assay is typically performed in a cylindrical reservoir with a round or conical bottom shape forming a constriction or throat. In the absence of agglutination, particles settle on the bottom of the reservoir and form a “button,” a region of high density particles. When agglutination occurs, the rigidity of the cross-linked particle complex prevents particles from settling and no button is observed on the bottom of the reservoir.
In practice, presence or absence of a button at the bottom of the reaction vessel is often determined by the unaided eye, a method that poses significant drawbacks. For example, this method requires careful assessment of each well and manual recordation of results, both of which are time consuming. Another example is that due to incomplete button formation (often referred to as a “halo effect”), the user routinely must tilt the entire plate at an angle and characterize the dynamic behavior of the incomplete button in order to interpret the data. Both of these examples of requirements in interpretation of the assay as well as others contribute to a significant level of subjectivity in interpretation of results, and variability in results between users.
The process of reading hemagglutination assay results is thus tedious, time-consuming, and requires user expertise and experience to reduce subjectivity and variability. These limitations restrict the number of personnel capable of providing reliable interpretation, and often result in plate reading being a bottleneck in the overall process of evaluating unknown samples. For a large company, central laboratory facility or organization, such as the US Centers for Disease Control and Prevention (“CDC”), that processes large numbers of unknown samples using agglutination assays, the manual analysis of agglutination assay results is a major limitation to throughput. Thus, an automated method for determining the presence or absence of agglutination is desirable.
Two main approaches have been previously described for the automated, optical detection of agglutination. In the first approach (direct approach), direct measurements of changes in the bulk optical properties of the reaction materials are made before and after cross-linking of particles to form clumps. A known or calibrated difference in an observed optical property of agglutinated and non-agglutinated reaction materials is used to detect the occurrence of agglutination. Examples of such optical properties include the tendency of the bulk reaction materials to scatter light, or the optical density of the bulk reaction materials. With this direct approach, a signal is generated in the case that agglutination occurs, and no significant signal is detected in the absence of agglutination. Examples of this approach are described in U.S. Pat. No. 4,829,011 to Biotrack, Inc. (1987), U.S. Pat. No. 4,760,030 to Syntex U.S.A., Inc. (1984), U.S. Pat. No. 3,819,271 to Max-Planck Gesellschaft Zur Forderung der Wissenschaften e.V. (1974), U.S. Pat. No. 4,597,944 to Cottingham (1986), U.S. Pat. No. 5,043,289 to Serres (1991), and U.S. Pat. No. 5,922,551 to Accumetrics, Inc. (1999).
Agglutination detection methods that employ the direct approach suffer from various drawbacks. For example, components of the agglutination reaction may absorb light in a wavelength-dependent manner, limiting the wavelengths of light that can be used to measure the optical properties of the reaction or limiting the media in which the agglutination experiment can be performed. As another example, non-specific agglutination of a small fraction of the particles in the reaction may cause significant changes in the bulk optical property being measured, leading to results that are false or difficult to interpret. As another example, in a scattering measurement the size of particles affects their scattering cross-section, thus limiting the size of particles or the wavelength of light that can be used in the assay. As another example, light absorption of some particles such as erythrocytes varies with oxygenation, causing changes in optical properties of the reaction in the absence of agglutination that may be misinterpreted as agglutination. Thus, while a few examples are provided, there are limitations to the utility of direct methods of optical detection of agglutination.
The second approach to optical detection of agglutination is indirect. After sufficient time for particles to have settled to the bottom of a reaction vessel, an image of the bottom of the vessel is formed, recorded and analyzed. In this method, a signal is generated in the absence of agglutination: particles that have settled in the absence of agglutination generate a pattern on the bottom of the reaction vessel that is detectable by analyzing the image of the bottom of the vessel.
Several examples of automated detection of agglutination by such indirect methods have been previously described, for example, in European patent application EP0588969A1 to Abbott Laboratories (1991), patent EP0198327 to Green Cross Corporation (1985), and U.S. Pat. No. 5,169,601 to Suzuki Motor Corp. (1990), U.S. Pat. No. 4,452,759 to Olympus Optical Company Ltd. (1980), U.S. Pat. No. 4,575,492 to Commissariat a l'Energie Atomique (1986), and U.S. Patent Application US2009/0325148 to Vaxdesign, Inc. (2008). While these methods describe various automated approaches to indirect detection of agglutination, they share a common element that the bottom of the reaction vessel is spatially imaged in at least one dimension to map the distribution of settled particles.
One disadvantage of these indirect optical methods is the requirement of considerable hardware such as focusing optics to create an image on an image plane or surface. Another disadvantage of these indirect optical methods is the requirement of either an array of light sensors for each well or a single sensor combined with mechanical hardware for scanning the single sensor across the bottom of each well to collect the image formed. This specialized imaging and image-collecting hardware adds to the cost and complexity of an instrument, and in the case of a scanning imaging system, increases the time required to evaluate agglutination in each well.
Another disadvantage associated with indirect measurement of the imaging type is that it is difficult to collect substantially continual measurements of the agglutination process with these methods. The time required to scan a sensor across the bottom of a well to form an image limits the time resolution of substantially continual measurements, thus limiting the potential utility of such measurements in detecting rapid changes during agglutination. The fixed sensor array method for collecting an image of the bottom of the reaction vessel to detect agglutination is limited by the time required to process the image at each time point, which either limits the time resolution of the data collection or requires a large amount of time after the assay is complete to process each image from the time series.
Another disadvantage associated with indirect measurement is that it requires imaging is that the approach is not easily amenable to making parallel measurements of multiple wells. The cost and complexity of the imaging optics and sensor array, as well as the difficulty of properly aligning such optics for each well prohibits large-array scaling of such methods.
The drawbacks of the conventional direct and indirect measuring methods makes improved optical detection methods desirous. The need for improvement is demonstrated in part as after nearly thirty years since the development of the above optical methods of agglutination detection: a single CDC laboratory will, for sustained periods, evaluate thousands of reaction vessels each day for agglutination in HA or HAI assays, and to do so they evaluate the samples using the unaided eye of highly experienced, trained scientists rather than one of the aforementioned detection methods. Against this background, the technology of the present application provides an automated method of detecting agglutination in an agglutination reaction.